This invention relates to the field of therapy and diagnostic methods for ulcerative colitis. Specifically, the method comprises administering a compound or recombinant protein that inhibits interaction between CEP and human tropomyosin. Also included in the invention are methods to screen for drugs useful in treating ulcerative colitis.
Various scientific and scholarly articles are referenced throughout the specification. These articles are incorporated by reference herein to describe the state of the art to which this invention pertains.
The Ca2+ dependence of vertebrate skeletal muscle contraction is due entirely to a set of specialized accessory proteins closely associated with actin filaments. If myosin is mixed with pure actin filaments in a test tube, myosin ATPase is activated whether or not Ca2+ is present; in a normal myofibril, on the other hand, where the actin filaments are associated with accessory proteins, the activation of the myosin ATPase depends on Ca2+.
One of these accessory proteins is a rigid rod-shaped molecule, called tropomyosin because of similarities to myosin in its x-ray diffraction pattern. Like the myosin tail, tropomyosin is a dimer of two identical xcex1-helical chains which wind around each other in a coiled coil. By binding along the length of an actin filament, tropomyosin stabilizes and stiffens the filament.
Tropomyosins are present in all eukaryotic cells. Different isoforms of tropomyosin, generated through alternative splicing, are expressed in a tissue-specific manner (Less-Miller, J P, et al., Bioassays 1991; 13: 429-37). In human fibroblast tissue, at least eight isoforms of TMs have been identified. These isoforms range in molecular weights from 30-40 kDa (Lin J J-C, et al., Int Rev Cytol 1997; 170:1-38). Classically, tropomyosins are known to remain intracellular because they lack the signal sequence required for membrane insertion and translocation (Less-Miller, supra).
Human tropomyosin (hTM) is a cytoskeletal microfilament protein. A significant number of ulcerative colitis patients show a preferential immune response to hTMs, in particular, the hTM5 isoform. Thus, hTM is a candidate autoantigen in ulcerative colitis. Using lamina propria lymphocytes from mucosa of patients with ulcerative colitis and ulcerative colitis sera, an autoantibody response to hTM isoforms has been demonstrated in several independent studies, including that of Das, K M, et al. J Immunol. 1993; 150:2487-93. Such an anti-hTM autoantibody response, however, was not seen in patients with Crohn""s disease. Recently, these findings were extended to an animal model of colitis using TCRxcex1xe2x88x92/xe2x88x92 mice (Mizoguchi, A., et al. J. Exp Med. 1996; 183: 847-56). Severity of colitis in these mice is directly correlated with the increased titer of anti-TM autoantibodies and the increased number of appendicular B cells producing anti-TM autoantibodies (Mizoguchi, A., et al. J. Exp Med. 1996; 184:707-15).
In colon epithelium, the most predominantly expressed hTM isoform is hTM5 (Geng X, et al., Gastroenterology 1998; 114:912-22). It is presently unknown whether hTM5 is accessible to anti-TM autoantibodies, particularly when the target protein is expected to be exclusively intracellular. The possibility of externalization of hTM5 in colon epithelium and likelihood of the passive transport of hTM5 with a secretory protein has been considered. One likely candidate for this chaperone function is a colon epithelial-specific protein recognized by the 7E12H12 monoclonal antibody.
The monoclonal antibody 7E12H12 was raised using highly enriched colonic tropomyosin (earlier named as 40 kDa protein or p40) (Das K M, et al., J. Immunol 1987; 139:77-84). However, 7E12H12 does not react with any of the known hTM isoforms in ELISA or immunotransblot analysis, either from muscle as well as from non-muscle epithelial cells (Das K M, et al., Gastroenterology 1997; 112:A955). However, the 7E12H12 monoclonal antibody recognizes a cell membrane associated protein present exclusively in the colon epithelium (Das K M, et al. (1987) supra; Das K M, et al. (1997) supra). By immunotransblot analyses, CEP has been identified as a high molecular weight ( greater than 200 kDa) protein present in colon epithelial cells but not in small intestinal enterocytes. Among the colon cancer cell lines, LS-180, and DLD-1 cells express the 7E12H12-reactive protein but HT-29 cells do not (Hassan T., et al., Clin Exp Immunol. 1995; 100:457-62).
In accordance with the present invention, it has been found that hTM5 is externalized in colon epithelium but not in small intestinal epithelium, despite the lack of a signal peptide. Furthermore, hTM5 is specifically associated with the colon epithelial-specific protein (CEP), and both are found to be secreted by LS-180 colon cancer cells.
The first aspect of the invention is due to the new appreciation that hTM is externalized in the colon epithelium and thus can stimulate the immune system and provide its antigenic role. The physical interaction of hTM with CEP is also now appreciated as important for the release of hTM outside the cell. Since an autoantibody response to hTM is associated with ulcerative colitis, the condition can be treated by decreasing the externalization of hTM in the colon. The first aspect of the invention is therefore a prophylactic and therapeutic method for treating or preventing ulcerative colitis and other diseases associated with an autoantigen response to hTM in patients in need of such a treatment. In preferred embodiments, the hTM isoform is hTM5. In a preferred embodiment, this treatment method comprises administering a compound to target cells. The compound inhibits the externalization of hTM and/or interaction between CEP and hTM within target cells. In preferred embodiments, the target cells are colon cells. In a more preferred embodiment, the compound inhibits the interaction between CEP and hTM by physically binding either within the cell. In a particularly preferred embodiment, the compound is a recombinant protein that has a functional hTM binding domain from CEP or CEP-like proteins and competes for hTM binding in vivo. In another preferred embodiment, the compound decreases or causes a decrease in the expression of the CEP protein in target cells. In another preferred embodiment, the compound reduces the release of hTM from colon cells. The compound may also prevent secretion of the CEP-hTM complex from the target cells. In a more preferred embodiment, the compound affects the organization of the cytoskeleton and/or inhibits active secretion. In a most preferred embodiment, the compound is phorbol-12-myristate-13-acetate, monensin or methylamine.
Another aspect of the invention is a prophylactic or therapeutic method to treat ulcerative colitis and other diseases associated with an autoantigen response to hTM that uses the specific binding of CEP to hTM to decrease or remove the autoantigenic nature of hTM. One embodiment of the method entails administering a recombinant protein that comprises a functional hTM binding site from CEP operably linked to a non-antigenic protein. Another embodiment of the method entails tolerization by repeated oral feeding of the hTM and/or CEP.
Another aspect of the invention is method to identify drugs that are useful for treating ulcerative colitis and other diseases associated with an autoantigen response to hTM which targets the disassociation of the CEP-hTM complex. In a preferred embodiment, intracellular association of CEP and hTM is determined by hTM secretion from human colon cells, with a decrease in hTM secretion indicative of a drug with therapeutic properties. In a more preferred embodiment, the LS-180 cells are used.
Another aspect of the invention is a diagnosis method for detecting diseases associated with an autoantigen response to hTM which entails detecting CEP-hTM complexes in affected tissue. Presence of CEP-hTM complexes are indicative of disease. In one embodiment, the complexes, or a part of them, are detected in the extracellular space of the affected tissue or by sensitized lymphocytes form colonic mucosa. In another embodiment, the CEP-hTM complexes are detected in intracellular space of the affected tissue. In a preferred embodiment, the tissue is the colon epithelium.
Other features and advantages of the present invention will be better understood by reference to the drawings, detailed description and example that follow.
The specific interaction between the colon epithelium-specific protein (CEP) and the autoantigen human tropomyosin (hTM) is known to be a fundamental element in the development of the inflammatory bowel disease ulcerative colitis. Formation of CEP-hTM complexes is a requirement for the secretion of hTM from the colon epithelium and the resulting autoimmune response. Secretion of hTM in the colon epithelium and not in the small intestine is due to expression of CEP in the colon epithelium. While not limiting the functioning of the invention to any one explanation, it is likely that the intracellular interaction of the secretory protein CEP and intracellular protein hTM allows for the passive transport of hTM with CEP out of the cell.
Thus, it is likely that the externalization of hTM5 is by cotransport of hTM5 with a secretory protein. The externalization of hTM5 in the colon epithelial cells and not in small intestinal enterocytes, suggests that a colon-specific factor is involved in the process. This is supported by the fact that the 7E12H12 monoclonal antibody was raised by injecting highly enriched colonic TM (previously identified as p40) (Das K M, et al. J Immunol 1987;139:77-84), and the correlation between the cell surface expression of colon-specific protein, CEP and hTM5. HTM5 is expressed intracellularly in both colonic and small intestinal epithelial cells. Yet, cell surface expression of hTM5 is seen only on colonic epithelial cells, and LS-180 cells which express CEP but not in small intestinal epithelial cells and HT-29 cells that lack CEP. This is further substantiated by the recovery of intact hTM5 and CEP in the culture medium of LS-180 cells. The coimmunoprecipitation experiments using anti-hTM5 monoclonal antibody and 7E12H12 monoclonal antibody of the present invention further demonstrates physical association of hTM5 with CEP. Disappearance of 31 kDa protein, not precipitable by LC1 after immunoprecipitation with 7E12H12 monoclonal antibody also suggests that CEP associated 31 kDa protein is hTM5.
CEP of the present invention is identified as a high molecular weight protein ( greater than 200 kDa). This was evidenced using colon epithelial cell and LS-180 colon cancer cell lysates as well as LS-180 culture medium by immunotransblot analysis with 7E12H12. Further, the monoclonal antibody, 7E12H12 did not react with any other proteins including the hTM isoforms 1-5 with molecular weights that range from 31 kDa to 40 kDa in any of the cell lysates or the culture medium that contain hTM.
CEP is a membrane associated protein that is secreted from the colon cells. hTM5 is associated with the cytoskeleton. CEP and hTM5 are believed to remain segregated in the cell. TM isoforms are involved in stabilizing actin filaments, changing cell shape, and regulating intracellular granule movement and cytokinesis (Lin J J-C, et al., (1997) supra). Actin cytoskeleton is known to be involved in the stimulus-dependent regulated exocytosis of stored secretory granules in LS-180 cells (McCool D J, et al. (1995) supra). Based on the observation that hTM5 is coimmunoprecipitated with CEP, at least a small amount of CEP may enter regulated exocytosis via stored secretory granules. This may facilitate the access of hTM5 to the secretory pathway.
The present invention demonstrates that two secretagogues, A23187 and PMA, have different effects on the secretion of CEP and hTM5. CEP secretion was unaffected in response to both A23187 and PMA. However, hTM5 secretion was increased by A23187 and inhibited by PMA. It is known that A23187 and PMA stimulate mucin secretion in LS-180 cells (McCool D J, et al. (1995) supra). However, PMA can affect the organization of cytoskeleton and thus may interfere with the secretion of hTM5 (Derventzi A., et al., Biochem Biophys Res Commun 1992; 182: 1423-28). The secretion of hTM5 may be inhibited by drugs known to inhibit Golgi-dependent and Golgi-independent secretion of proteins, such as monensin, and methylamine, respectively. Although the secretory pathway of hTM5 in LS-180 cells is not known, the inhibitory effect of these two drugs further supports the hypothesis that hTM5 is actively secreted by LS-180 cells. Cell surface expression of CEP is sensitive to monensin, suggesting the involvement of Golgi in the transport of CEP molecules. Thus, the present invention demonstrates that a Ca+2 ionophore such as (A23187) facilitates the interaction between CEP and hTM5, whereas PMA, monensin, and methylamine interfere either with the physical interaction or cotransport of the CEP-hTM5 complex.
Cell surface expression and secretion of hTM5 are relevant to the autoimmune mechanisms in ulcerative colitis because the auto antibody response is directed to colonic tropomyosins. Colonic tropomyosins may also be available for the mucosal immune system during apoptosis of epithelial cells. A physico-chemical analysis of common structural motifs present in 109 human auto antigens has revealed that tropomyosins have the highest number of such motifs and thus, a very high propensity to act as auto antigens (Dohlman J G, et al., Biochem Biophys Res Commun 1993; 195: 686-96). The putative target antigen for autoantibodies in ulcerative colitis, such as hTM5 or hTM related peptide, may stimulate the effector immune system and can promote destruction of the epithelium by various immune mechanisms, including complement activation. Autoantibodies to the intracellular proteins are found in many autoimmune diseases and in most cases, such antibodies are considered nonpathogenic unless the target auto antigen is accessible to the antibodies. Several studies demonstrated that autoantibodies can also be internalized inside the target cells and may cause cell damage (Alarcon-Segovia D., et al., Immunol Today 1996; 17:163-64). The possibility that the intracellular target antigens are exposed to the autoantibodies is demonstrated in a mouse model for experimental autoimmune myocarditis as reported by Lio L., et al., J. Exp Med 1995; 181: 1123-31. In this model, autoantibodies to myosin cause tissue damage and inflammation, presumably via complement fixation.
The present invention indicates that CEP functions as a chaperone for the transport of hTM5. The lack of CEP in the small intestine can explain why hTM5 is not externalized by small intestinal enterocytes. This may also be relevant as to why ulcerative colitis is restricted to the colon. In the gastrointestinal tract, CEP is expressed only in colon epithelial cells. However, at extracolonic sites, CEP is expressed in skin and biliary epithelium, ciliary epithelium in eye, and also in chondrocytes. These are organs and tissues commonly involved in inflammatory bowel disease. As shown by immunoperoxidase studies using fixed formalin tissue, hTM5 is also expressed at these extraintestinal sites (Marks M, et al., Gastroenterology, 1998; 114:A1032. Thus, the present invention suggests a possible role for the CEP+hTM5 complex in the immunopathologenesis of ulcerative colitis.
The association of the CEP and hTM proteins forms the basis of the present invention relating to ulcerative colitis and associated diseases. The present invention also includes prophylactic and therapeutic methods to treat ulcerative colitis as well as associated disorders that involve disturbing the binding of CEP to hTM in order to remove the hTM autoantigen from the extracellular space. Also contemplated in the present invention are aspects that use the presence of the CEP-hTM complex in diagnostic methods for ulcerative colitis and related disorders. The present invention also includes a method to identify drugs that will be helpful for the treatment of ulcerative colitis and related diseases.
The compounds of the present invention may be any compound associated with ulcerative colitis that inhibits the externalization of hTM from colon epithelial cells. Preferably, the compound inhibits the formation of the CEP-hTM complex in a target cell. More preferably, the compound inhibits the interaction between CEP and hTM5 by physically binding to either CEP or hTM5 within the target cell. Even more preferably, the compound is a recombinant protein that acts as a functional hTM binding site from CEP. Preferably, administration of the inventive compound preferably results in a decreased expression of the CEP protein in target cells, and/or prevents secretion of the CEP-hTM complex. This inhibition of CEP-hTM complex may be achieved by affecting either the cytoskeletal organization of the cell or active secretion. Examples of suitable compounds for the above mentioned purposes are phorbol-12-myristate-13-acetate, monensin and methylamine.
The amount of compound that is administered is a therapeutically effective amount. The exact amount of compound used is a matter of preference subject to such factors as the type of condition being treated (e.g., ulcerative colitis) as well as the dosage recommended or permitted for the particular compound. In general, the amount of compound employed is the dosage required to obtain the desired result.
The present invention also extends to methods of treating or preventing ulcerative colitis and other associated diseases comprising administering a compound of the invention to a target cell. If the compound is a recombinant protein, the recombinant protein ideally comprises a functional hTM binding site found on the CEP protein. This binding site is operably linked to a non-antigenic protein.
The compounds and recombinant proteins of the present invention may be administered together with pharmaceutically acceptable carriers to provide pharmaceutical compositions which can be administered to a human orally or rectally or both, in amounts effective to provide a variety of therapeutic activity. Of course, the type of carrier will depend on the mode of administration desired for the pharmaceutical composition as is conventional in the art as well as the desired site of action. Preferably, the compound or protein is administered orally or rectally to the human.
It is especially advantageous to formulate the pharmaceutical compositions in dosage unit forms for ease of administration and uniformity of dosage. The term, xe2x80x9cdosage unit formsxe2x80x9d as used herein refers to physically discrete units suitable for use as a unitary dosage, each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the pharmaceutical carrier.
Extraintestinal manifestations in patients with inflammatory bowel disease are very common. The majority of these manifestations accompany the underlying disease and are influenced by its activity. Manifestations include those listed in Table 1. Because these extraintestinal manifestations are closely associated with inflammatory bowel disease, it is likely that many share their origin in the autoantigenic properties of externalized hTM. The diagnosis and treatment of these extraintestinal disorders are also contemplated with regards to the methods of the present invention.
Thus, also included in the present invention are methods of screening for drugs that are useful in treating ulcerative colitis and associated diseases. One such method of screening comprises administering a candidate drug to human colon cancer cells and subsequently determining the amount of CEP-hTM complex in the cells. The amount of CEP-hTM complex is compared to either a negative control (e.g., a cell not treated with the putative drug) or to levels of CEP-hTM complex measured before administration of the putative drug. A decrease in CEP-hTM complex levels are indicative of therapeutic value of the drug. In one embodiment, this cell based assay measures, the amount of CEP-hTM complex is determined by quantifying the amount of hTM secreted from the colon cancer cells. In a highly preferred embodiment, the colon cancer cells are LS-180 cells. Detection of secreted hTM may be achieved using Western analysis immunoprecipitation as described in Example 5, below, on the serum/culture medium with an antibody specific for the secreted hTM.
Another aspect of the invention includes a cell-free assay for binding CEP-hTM complexes. The hTM and CEP proteins are combined in a cell-free system and contacted with the test compound. The cell-free system is selected from a group consisting of a cell lysate and a reconstituted protein mixture. The hTM and CEP are simultaneously expressed in a cell, and the cell is contacted with the test compound. Suitability of the test compound is determined by measuring the ability of the test compound to cause a decrease in hTM-CEP complex formation. Detection of hTM-CEP complexes may be achieved using conventional techniques including but not limited to gel electrophoresis or size exclusion gel chromatography. These methods are routine and well-known to one of ordinary skill in the art.
Also included in the present invention are expression based assays comprising a CEP gene and a promoter to form a construct, wherein the construct is operably linked to a reporter gene.
The present invention is not limited to the embodiments described and exemplified above, but is capable of variation and modification without departure from the scope of the appended claims.
Definitions
As used herein, the term, xe2x80x9ctreatingxe2x80x9d includes a therapeutic treatment of an existing or established disorder in which CEP binding to hTM is affected, or prevention of the symptoms in a subject at risk for a disorder in which CEP-hTM interaction is affected.
For purposes of the present invention, the term, xe2x80x9csubjectxe2x80x9d is intended to include human and non-human animals. The term, xe2x80x9cnon-human animalsxe2x80x9d includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dog, cow, chickens, amphibians, reptiles, etc. In certain embodiments, the subject is a mammal, e.g., a primate, e.g., a human.
In the present invention, the term, xe2x80x9cadministeringxe2x80x9d is intended to include routes of introducing to a subject at least one compound to perform its intended function. Administration can be carried out by any suitable route of administration, including orally, intramuscularly, or intraperitoneal.
As used herein, the term, xe2x80x9ctherapeutically effective amountxe2x80x9d refers to the quantity of compound necessary to prevent, to cure or at least partially arrest the symptoms of the disease or disorder and its complications.
By xe2x80x9cbindingxe2x80x9d is meant a physical association between two or more molecules which is more prolonged and/or of greater strength or affinity than would be observed following random collisions of molecules that do not bind to one another. Such binding may be transient or for a longer period.
By xe2x80x9cscreeningxe2x80x9d is meant a process in which a large number of potentially useful agents are processed in the method of this invention. It is a process distinct from a single experiment in which a single agent is studied in detail to determine its method or mode of action.
By easily detectable reporter is meant any agent or substance which can be readily detected by physical, chemical, biochemical, enzymatic or other means. Such reporters include, but are not limited to, enzymes, fluorescent, luminescent, or chromophoric molecules, antibodies labeled with any of the foregoing, and haptens and antigens that can be detected using such antibodies.
By xe2x80x9cin vitro translation systemxe2x80x9d is meant a cell-free extract capable of translating a protein or proteins from an RNA or RNAs encoding such proteins. Such a mixture typically contains ribosomes, tRNAs, amino acids, salts, and various other factors required to sustain protein synthesis, in addition to the RNA(s) that direct protein synthesis. Such mixtures are typically prepared from sources such as, but not limited to, rabbit reticulocytes, HeLa cells, wheat germ, and E. coli cells; extracts prepared from such sources may be supplemented by the addition of tRNAs, amino acids, and so on, as necessary.
The term xe2x80x9coperably linkedxe2x80x9d or xe2x80x9coperably insertedxe2x80x9d means that the regulatory sequences necessary for expression of the coding sequence are placed in a nucleic acid molecule in the appropriate positions relative to the coding sequence so as to enable expression of the coding sequence. This same definition is sometimes applied to the arrangement other transcription control elements (e.g. enhancers) in an expression vector.
Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, polyadenylation signals, terminators, and the like, that provide for the expression of a coding sequence in a host cell.
The terms xe2x80x9cpromoterxe2x80x9d, xe2x80x9cpromoter regionxe2x80x9d or xe2x80x9cpromoter sequencexe2x80x9d refer generally to transcriptional regulatory regions of a gene, which may be found at the 5xe2x80x2 or 3xe2x80x2 side of the coding region, or within the coding region, or within introns. Typically, a promoter is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3xe2x80x2 direction) coding sequence. The typical 5xe2x80x2 promoter sequence is bounded at its 3xe2x80x2 terminus by the transcription initiation site and extends upstream (5xe2x80x2 direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence is a transcription initiation site (conveniently defined by mapping with nuclease S1), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.